CALD1 inhibits invasion of human ovarian cancer cells by affecting cytoskeletal structure and the number of focal adhesion

Abstract

Background: Ovarian cancer (OV) is associated with the highest mortality rate among gynecological cancers, largely due to late diagnosis and chemoresistance. The identification of novel diagnostic markers and therapeutic targets is crucial. Caldesmon 1 (CALD1), a cytoskeleton-regulating protein, has been implicated in various cancers. This study aims to investigate the expression and functional significance of CALD1 in OV, focusing on its potential impact on cell invasion and metastasis.

Methods: We analyzed CALD1 expression using The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases, along with tissue microarray immunohistochemistry (IHC). Drug sensitivity analysis was performed using the 'oncopredict' R package. A CALD1 gene network was constructed, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. SK-OV-3 cell lines with stable CALD1 knockdown were established and verified by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot (WB). We then assessed cell invasiveness using Transwell assays and visualized cytoskeletal changes through immunofluorescence staining of F-actin and Vinculin.

Results: The expression of CALD1 was significantly reduced in OV tissues compared to normal tissues. Patients with high and low expression levels of CALD1 showed significant differences in their response to chemotherapeutic drugs. CALD1 and its related genes were found to play an essential role in regulating cytoskeleton organization, focal adhesion formation, and cell movement processes. CALD1 knockdown cells exhibited a significant reduction in F-actin stress fibers, a loose cytoskeleton structure, decreased Vinculin expression, and enhanced migration ability.

Conclusions: Attenuated expression of CALD1 in SK-OV-3 cells leads to fewer F-actin stress fibers, reducing the association between the cytoskeleton and Vinculin. This results in reduced cellular focal adhesions and increased invasiveness of SK-OV-3 cells, promoting OV cell metastasis. These findings suggest that CALD1 may have important clinical implications in the diagnosis and treatment of OV.

Keywords: Caldesmon 1 (CALD1); Ovarian cancer (OV); cytoskeleton; focal adhesion.

Figure 1

Differential analysis of CALD1 expression in tumor tissue and healthy tissue. (A) Analysis of CALD1 expression levels in ovarian cancer and normal tissues from TCGA and GTEx databases. (B) Comparison of the differences in CALD1 expression between ovarian cancer tissues and normal tissues on tissue microarrays. (C,D) Representative image of immunohistochemistry of normal tissue on tissue microarray. (E,F) Representative image of immunohistochemistry of ovarian cancer tissue on tissue microarray. Scale bar: 200 µm. ****, P<0.0001. TPM, transcripts per million; OV, ovarian cancer; H-Score, Histochemistry Score; TCGA, The Cancer Genome Atlas; GTEx, Genotype-Tissue Expression.

Figure 2

Drug sensitivity analysis of high and low CALD1 expression groups. This figure presents a drug sensitivity analysis for groups with high and low CALD1 expression, performed using the ‘oncopredict’ R package. Each boxplot illustrates the sensitivity differences for a specific drug between the high expression group (red) and the low expression group (blue). The y-axis represents drug sensitivity, while the x-axis categorizes the groups based on CALD1 expression levels. The P values above each boxplot indicate the statistical significance of the differences observed between the two groups. Drugs analyzed include irinotecan, oxaliplatin, topotecan, CDK9_5038, entospletinib, AZD5363, AZD5991, LCL161, GNE-317, tozasertib, entinostat, and telomerase inhibitor IX.

Figure 3

Construction and enrichment analysis of CALD1 gene network. (A) Construction of CALD1 gene network using GeneMANIA. If two genes have a relationship, such as co-expression, shared protein domain, physical interactions, co-localization, or pathway, connecting lines are drawn. The thickness of the lines represents the degree to which two genes are similar. (B,C) GO enrichment analysis and KEGG enrichment analysis. Bar chart of GO and KEGG enrichment analysis results sorted by P value. GO, Gene Ontology; GeneMANIA, Gene Multiple Association Network Integration Algorithm; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 4

Screening and construction of cell lines with stable knockdown of CALD1. (A) Fluorescence photomicrographs of SK-OV-3 cells 72 h after lentivirus infection. (B,C) Knockdown of CALD1 in SK-OV-3 cell lines analyzed by qRT-PCR and WB. One-way ANOVA was used for statistical analysis. *, P<0.05; **, P<0.01. NC, negative control group; qRT-PCR, quantitative real-time polymerase chain reaction; WB, western blot; ANOVA, analysis of variance.

Figure 5

Transwell invasion assay. (A) Representative images of Transwell invasion assays for different SK-OV-3 cell groups. (B) Results analysis of Transwell assay. One-way ANOVA was used for statistical analysis. ***, P<0.001. OD, optical density; NC, negative control group; ANOVA, analysis of variance.

Figure 6

F-actin was visualized by staining with rhodamine-phalloidin (red). Nuclei were stained with DAPI (blue). Scale bar =25 µm. (A) Representative images of actin cytoskeleton staining for different SK-OV-3 cell groups. The right-side images are approximately two times magnified compared to the corresponding areas in the left images. (B,C) The fluorescence intensity of the cytoskeleton and focal adhesion (Vinculin) was quantified using ImageJ software. One-way ANOVA was used for statistical analysis. **, P<0.01; ***, P<0.001; ****, P<0.0001. ANOVA, analysis of variance.

Figure 7

Immunofluorescence staining images of the focal adhesion marker Vinculin (red) in different SK-OV-3 cell groups. Nuclei were stained using DAPI (blue). The right-side images are approximately two times magnified compared to the corresponding areas in the left images.